The flexibility of myosin 7a

Myosin 7a is a molecular motor found in hair cells of the ear and the photoreceptor cells of the eye. Myosin 7a is comprised of an actin-binding motor domain, a lever; which is composed of 5 IQ motifs that can potentially bind 5 light chains followed by a single alpha helical (SAH) domain, and a tail composed of 2 MyTH4-FERM domains. The lever is an essential mechanical element in myosin 7a function, but an understanding of its mechanical properties and how these derive from its substructure is lacking. It has been observed in vitro that myosin 7a is able to regulate its activity through a head-tail interaction. How the flexibility of the sub-domains of the lever allows the molecule to fold up is not completely understood. To address this, the first aim of this study was to look for evidence of novel light chain binding partners in myosin 7a, which revealed calmodulin to be the preferred light chain. My second aim was to study the structure and flexibility of the lever of full-length myosin 7a using single-particle image processing of images from negative stain electron microscopy (EM). Image averaging revealed the lever to be much shorter than expected. Additionally, there was evidence of thermally-driven flexing at the motor-lever junction. A stiffness of 78 pN.nm.rad-2 for the flexing was inferred, which represents a significant compliance in the head. An investigation into lever bending analysis, by monitoring the decay of tangent-tangent correlations of the lever shapes, yielded a persistence length of 38 ± 3 nm. Finally, long time molecular dynamics (MD) simulations were compared with a novel coarse-grained (CG) simulation technique called Fluctuating Finite Element Analysis (FFEA), which treats proteins as visco-elastic continua subject to thermal noise to probe the flexibility of myosin 7a. FFEA allows sufficiently long time simulations that are computationally less expensive than corresponding all-atom MD simulations to allow myosin 7a to explore its full range of configurations. Extraction of flexibility data from all-atom MD simulations calculated the bending stiffness of the SAH domain to be 60.5 pN.nm2, with reasonable overlap of the major modes of motion between the all-atom and CG simulation types